An ENCODE ChIP-seq pipeline using endogenously tagged human DNA-associated proteins

使用内源标记的人类 DNA 相关蛋白的 ENCODE ChIP-seq 流程

• 批准号: 10240992
• 负责人: Eric Mendenhall
• 金额: $ 94.31万
• 依托单位: HUDSON-ALPHA INSTITUTE FOR BIOTECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10240992
• 关键词: Alleles; Antibodies; Basic Science; Biological Assay; Biological Process; Biology; Cell Nucleus; Cells; ChIP-seq; Chromatin; Chromosomes; Clinical Research; Code; Communities; DNA; Data; Development; Elements; Encyclopedia of DNA Elements; Environment; Epitopes; Failure; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Goals; High-Throughput Nucleotide Sequencing; Human; Human Biology; Human Cell Line; Human Genome; Location; Maps; Molecular; Mus; Mutation; Pathogenicity; Pathologic; Phase; Physiological; Plasmids; Production; Proteins; Research; Technology; Testing; Transcriptional Regulation; cell type; chromatin immunoprecipitation; genetic makeup; genetic regulatory protein; genome wide association study; genome-wide; human DNA; human disease; improved; novel strategies; response; transcription factor

Project Summary/Abstract Almost a tenth of human genes code for proteins that interact with chromosomes in the nucleus. Most of these DNA-associated proteins (referred to as DAPs) are involved in regulating gene expression, by serving as part of the basic transcriptional machinery, as transcription factors that regulate the spatial and temporal levels of transcription, or as chromatin state regulators. These proteins are key components in biology, as transcriptional regulation underlies fundamental biological processes in organismal development, in determining cell states during differentiation, and in directing physiological responses to the internal and external environment. Thus, comprehensive and detailed assessment of the molecular actions of DAPs, that is, where they interact throughout the human genome, is a fundamental long-term goal of both basic and clinical research. In response to RFA-HG-16-002, “Expanding the Encyclopedia of DNA Elements (ENCODE) in the Human and Mouse (UM1)”, this application proposes to use a recently-established "shovel ready" pipeline for mapping DAPs in human cell lines that overcomes the very high failure rates of traditional ChIP-seq, a widely- used approach that requires specific antibodies for each factor. The new approach, called CETCh-seq, involves adding an epitope tag at the endogenous locus encoding each protein, and using chromatin immunoprecipitation with a universal antibody against the epitope followed by high-throughput sequencing (ChIP-seq) to identify DAP-DNA associations genome-wide. This production pipeline will be applied to each of 1,244 DAPs that are expressed in a set of human cell lines and have not yet been mapped by ENCODE. During the four-year project, this pipeline will be used to test each of these factors in one human cell line, and for 100 of the DAPs, in four human cell lines, allowing characterization of cell-type differences. The project will also tag and assay multiple allelic versions of a small number of DAPs in which pathogenic or potentially pathogenic mutations have been identified. The project will produce genome-wide DAP maps and identify motifs for hundreds of human regulatory proteins, providing an important component for the next phase of the ENCODE Project. All data, as well as useful materials in the form of gene editing plasmids and tagged human cell lines, will be made freely available to the research community.

项目总结/文摘

项目成果

Characterization of human transcription factor function and patterns of gene regulation in HepG2 cells.

• DOI: 10.1101/gr.278205.123
• 发表时间: 2023-12-01
• 期刊: GENOME RESEARCH
• 影响因子: 7
• 作者: Moyers, Belle A.;Partridge, E. Christopher;Mackiewicz, Mark;Betti, Michael J.;Darji, Roshan;Meadows, Sarah K.;Newberry, Kimberly M.;Brandsmeier, Laurel A.;Wold, Barbara J.;Mendenhall, Eric M.;Myers, Richard M.
• 通讯作者: Myers, Richard M.

A toolbox of immunoprecipitation-grade monoclonal antibodies to human transcription factors.

免疫沉淀级单克隆抗体的工具箱。

• DOI: 10.1038/nmeth.4632
• 发表时间: 2018-05
• 期刊: Nature methods
• 影响因子: 48
• 作者: Venkataraman A;Yang K;Irizarry J;Mackiewicz M;Mita P;Kuang Z;Xue L;Ghosh D;Liu S;Ramos P;Hu S;Bayron Kain D;Keegan S;Saul R;Colantonio S;Zhang H;Behn FP;Song G;Albino E;Asencio L;Ramos L;Lugo L;Morell G;Rivera J;Ruiz K;Almodovar R;Nazario L;Murphy K;Vargas I;Rivera-Pacheco ZA;Rosa C;Vargas M;McDade J;Clark BS;Yoo S;Khambadkone SG;de Melo J;Stevanovic M;Jiang L;Li Y;Yap WY;Jones B;Tandon A;Campbell E;Montelione GT;Anderson S;Myers RM;Boeke JD;Fenyö D;Whiteley G;Bader JS;Pino I;Eichinger DJ;Zhu H;Blackshaw S
• 通讯作者: Blackshaw S

Effects of sheared chromatin length on ChIP-seq quality and sensitivity.

剪切染色质长度对ChIP-seq质量和灵敏度的影响。

• DOI: 10.1093/g3journal/jkab101
• 发表时间: 2021-06-17
• 期刊: G3 (Bethesda, Md.)
• 作者: Keller CA;Wixom AQ;Heuston EF;Giardine B;Hsiung CC;Long MR;Miller A;Anderson SM;Cockburn A;Blobel GA;Bodine DM;Hardison RC
• 通讯作者: Hardison RC